ABSTRACT
Background: Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK [rDgK] antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test
Methods: The rDgK coding sequence was successfully sequenced, codon optimized and cloned in pET-24a[+] expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni[+2] - charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays [ELISA] with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test
Results: Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement [0.764] with molecular identification
Conclusions: The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test
ABSTRACT
Background: We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J [H2k] and BALB/c mice [H2d]. We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens [GRA2] combined with monophosphorryl lipid A [MPL] adjuvant elicits protective immune response against T. gondii
Methods: C57BL/6 [H2b haplotype] mice were immunized with GRA2, formulated in MPL adjuvant
Results: Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-gamma, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly [p < 0.01] fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-gamma production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization
Conclusions: We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii
ABSTRACT
Methylenetetrahydrofolate reductase [MTHFR] enzyme is one of the most important enzymes with a pivotal role in the folate metabolism and DNA synthesis pathways. Single nucleotide polymorphism [SNPs] in the coding gene has been related to many medical diseases as well as diverse malignancies including the prostate cancer which is the leading cause of the cancer deaths in men and one of the major public health problems. The goal of this study is to determine the relationship between the MTHFR C677T SNP and the prostate adenocarcinoma in Iranian males attending to the Labbafi-nezhad hospital in Tehran. In this Case-control unmatched study, 67 and 75 paraffinized tissue samples were taken out of the specimens diagnosed previously as the prostatic adenocarcinoma and nodular prostatic hyperplasia for the case and control groups respectively. MTHFR C677T genotyping was done by the use of multiplex ARMS-PCR and frequencies of the alleles were compared between the case and control groups as well as calculating the deviation from Hardy-Weinberg equilibrium and Odds Ratio for the "T" allele regarding the prostatic carcinoma. The observed rates in the control group were not too different from that of expected from Hardy- Weinberg equilibrium [P=0.407]. Frequencies of the possible genotypes were as follows: CC, 43.28% vs. 42.67%; CT, 49.25% vs. 52% and CT, 7.46% vs. 5.33% in the case and control groups respectively [P=0.85]. 1.37 times increased risk was found for the homozygote carriers of C677T variant [OR: 1.37, 95% CI: 0.33- 5.6; P=0.653] which is however statistically not significant. No association has been evident between the MTHFR 677C>T polymorphism and the risk of prostatic carcinoma in this study confirming the findings of some of the previous attempts; however, [OR: 1.37, 95% CI: 0.33-5.6] implies a slight effect of the homozygote on the carcinogenesis. Thus larger studies especially with a greater number of the smaples are recommended
ABSTRACT
Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies
ABSTRACT
Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii [T.gondii] throughout the world. Although usually asymptomatic, the infection can cause serious medical problems in immunocompromised individuals and fetuses. Toxoplasmosis also causes considerable economic loss because of abortion in livestock. DNA vaccination is a promising approach against intracellular parasites such as T.gondii. The goal of this study was to construct and evaluate functionality of a mammalian plasmid expressing GRA5 antigen of T.gondii as a possible DNA vaccine. GRA5 gene fragment devoid of the signal sequence, was amplified from genomic DNA of T.gondii RH strain, and cloned into pcDNA3.1 plasmid. The pcDNA3.1-GRA5 [pGRA5] was analyzed by restriction enzyme digestion followed by sequence determination. The pGRA5 was transfected into HEK 239-T human kidney cells, and expression of GRA5 antigen was investigated by Western blotting and immunofluorescence staining. The sequence encoding GRA5 was cloned into pcDNA3.1 plasmid. Restriction digestion of pGRA5 with Pst I enzyme showed correct in-sertion of GRA5 DNA into the plasmid. Sequence analysis revealed 100% homology with the published sequence of gra5. immunofluorescence and Western blotting analyses of HEK 293-T cells transfected with pGRA5 showed specific expression of GRA5. Immunogenicity of pGRA5 will be evaluated in mice
ABSTRACT
Diagnosis of Toxoplasma gondii [T.gondii] infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several tToxoplasma antigens, including dense granule antigens [GRAs] has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 [GRA8], excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli. [E.coli]. GRA8 was purified using an optimized single-step Iimmobilized Mmetal ion Aaffinity Cchromatography [IMAC]. The purity and yield of GRA8 was highest at pH= 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute tToxoplasma infection in pregnant women, an indirect immunoglobulin M [IgM] enzyme-linked immunosorbent assay [ELISA] was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection
Subject(s)
Humans , Female , Antigens, Protozoan , Escherichia coli , Toxoplasmosis/diagnosis , Pregnancy , Immunocompromised Host , Gene Expression , Immunoglobulin M , Rabbits , Enzyme-Linked Immunosorbent AssayABSTRACT
HTLV1 is the first detected retrovirus causing disease in human. The physiopathology of HTLV1 related diseases was mainly linked with its Tax protein characteristics. Use of mutant Tax proteins accompanied by immune regulator drugs could help treating HTLV1 associated myelopathy patients as a modulator of potent immune response against this viral protein. Since Tax protein is not commercially available, production of recombinant Tax protein was targeted for this study. Coding sequence of Tax protein [containing R222K mutation] in the pcDNA3.1[+] was digested with BamHI and XhoI restriction enzymes, and then removed and inserted into the expression vector pET32a[+] within the same cutting sites and cloned into E.coli DH5alpha. Recombinant vector was confirmed with enzymatic digestion, colony PCR, and sequencing of cloned gene. E.coli Rosetta [DE3] was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and western blot using monoclonal antibodies against Tax and 5His epitope. Finally, antigenic characteristic of the recombinant protein was evaluated by western blotting against patient sera. Presence of Tax protein band in the SDS-PAGE and western blot was confirmed. Western blotting of the recombinant protein with patient sera showed the band related to Tax protein. The recombinant protein is well produced and could be detected by patients' sera, making it eligible to be used as a recombinant viral antigen for future purposes
ABSTRACT
Dense granule antigens [GRA antigens] of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. GRA8 complementary DNA [cDNA], encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b[+]. Expression of recombinant GRA8 [rGRA8] was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. The cloned gene fragment exhibited complete similarity with the published sequence of GRA8 gene by sequence analysis. rGRAS was expressed in Escherichia coli infusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRAS recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRAS in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. The full length soluble rGRAS was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments
Subject(s)
Humans , Female , Animals, Laboratory , Antigens, Protozoan , Protozoan Proteins , Escherichia coli , Mice , RNA , Polymerase Chain Reaction , DNA, Complementary , Blotting, WesternABSTRACT
Toxoplasmosis is a worldwide infection which is commonly asymptomatic but can cause serious medical problems in immunocompromised individuals and fetus. The infection also causes considerable economic loss because of abortion in livestock, mostly in sheep and goats. DNA vaccination may be a powerful approach against intracellular parasites such as Toxoplasma gondii. The goal of this study was to construct and evaluate the functionality of an eukaryotic expression plasmid pRC/CMV-GRA2, harboring dense granule antigen-2 [GRA2] gene of T. gondii and to perform preliminary studies on its immunogenicity in a mouse model. The GRA2 complete cDNA was inserted in PCR2.1 plasmid, sequenced, then cut and inserted in pRC/CMV plasmid, to produce the recombinant plasmid pRC/CMV-GRA2 [pGRA2]. To verify that the plasmid construct pGRA2 was capable of expressing GRA2 in mammalian cells, it was transfected into 293-T cells, an embryonic kidney cell line. Western-blot analysis of the transfected cells using a monoclonal antibody specific for GRA2 indicated specific expression of GRA2 protein. CBA/J mice were subcutaneously immunized three times with 100 micro g of pGRA2 plasmid. The obtained sera recognized GRA2 that is shown by Western-blotting. These findings indicate that pGRA2 plasmid directs high-level expression of antigenic GRA2 protein in mammalian cells and is immunogenic in CBA/J mice